HSV is an enveloped, icosahedral, double-stranded DNA virus that infects mammals, including humans. Wild-type HSV infects and replicates in both terminally differentiated cells and dividing cells. Wild type HSV is neurovirulent, entering the peripheral nervous system where active viral replication is suppressed and the virus remains latent in neurones. HSV can reactivate from the latent state to produce infectious lesions. The HSV neurovirulence gene ICP34.5 is believed to condition post-mitotic cells (particularly neurones) for viral replication. (Thompson et al. Virology 172, 435-450 (1989); MacLean et al., J. Gen Virol. 72, 631-639 (1991); Harland and Brown, J. Gen Virol. 66, 1305-1321 (1985); and Taha et al., J. Gen Virol. 70, 705-716 (1989)). ICP34.5 deletion mutants cannot replicate in terminally differentiated cells but can lytically infect dividing cells (Brown, S. M et al. 75, 2367-2377 (1994)). The HSV-1 mutant strain 1716 is an ICP34.5 deletion mutant (EP-B-O 571,410) of HSV type I strain 17, that has reduced virulence and greatly reduced lethality in mice, but it replicates as efficiently as wild type virus in actively dividing tissue culture cells (Maclean, A. R. et al. J. Gen Virol. 72 631-639 (1991), Brown, S. M. et al, J. Gen Viral. 75 2367-2377 (1994)). The ability of ICP34.5 deletion mutants to specifically target and lyse dividing cells and not post mitotic cells makes them an attractive therapeutic agent for the treatment of cancer. HSV infection of rapidly dividing cancer cells leads to death of the cells by lysis. The 1716 mutant kills tumour cell lines in tissue culture and, in a range of in vivo cancer models, the virus was shown to induce tumour regression and prolong survival (Kesari, S., Randazzo, B. P. and Valyi-Nagy, T. Lab Invest. 73 636-648 (1995), MacKie E. A. et al. Br J. Cancer 74 745-752 (1996), Randazzo, B. P. et al. J. Invest Dermatol. 108 933-937 (1997)). In clinical trials, direct injection of 1716 was effective in treating patients with recurrent glioma (Rampling, R. et al. Gene Therapy 7 (10) 859-866 (2000)) and metastatic melanoma (MacKie, R. M., Stewart, B. and Brown, S. M. The Lancet 357 525-526 (2001)).
Significantly, in both of these trials there was no evidence for spread of 1716 to surrounding normal tissue. A second HSV-1 ICP34.5 deletion mutant, G207, which additionally lacks the UL39 gene that encodes the large subunit of the viral ribonucleotide reductase, has also been shown to be safe and effective in patients with malignant glioma (Markert, J. M. et al. Gene Therapy 7 (10), 867-874 (2000)).
Although strains such as 1716 and G207 that have impaired neurovirulence and that selectively infect dividing cells have strong therapeutic potential for the treatment of human malignancies, some limitations of their use are anticipated by the inventors. 1716 is able to infect and kill a variety of tumour cells in tissue culture, but its permissive range in vivo may be more restricted. For example, 1716 infection of B or T cell lymphomas has not been reported. Additionally, the virus may infect tumour cells less efficiently in vivo than in cell culture. The inventors have appreciated that it is desirable to overcome certain cell-type restrictions and increase the efficiency of infection of tumour cells so that modified HSV can be more widely, effectively and safely applied as an in vivo therapy.
Broadly, the present invention provides a means of altering or modifying the tropism of HSV, so that a particular range of cell types can be targeted.
At its most general, the present invention provides an HSV complex comprising a modified HSV and a targeting agent capable of targeting said modified HSV to a specific cell type, preferably a proliferating cell e.g. a cancer cell. The invention further provides a method of producing the HSV complex and its use of the complex in the treatment of diseases such as cancer.
Thus, in a first aspect, the present invention provides an HSV complex comprising an HSV linked to a targeting agent, capable of targeting a specific cell type, where the genome of said HSV is modified in the terminal portion of RL within Bam HI s (0-0.02 and 0.81-0.83 mu).
The targeting agent is conveniently an antibody or component of an antibody, e.g. an antibody binding domain. The antibody is preferably capable of specifically binding to a cell surface protein present of the cell type targeted. This is discussed below. The targeting agent is preferably linked to the modified virus via a viral envelope protein so that it is displayed on the surface of the virus. A convenient way of achieving this is to form a fusion protein comprising the targeting agent and a viral envelope protein such as a glycoprotein. Alternatively, the targeting agent may be linked to the viral particle by chemical means, e.g. co-valently, or by a binding agent, e.g. avidin/strepavidin and biotin.
In a preferred embodiment, nucleic acid encoding the targeting agent is incorporated into the viral genome so that it is expressed as a fusion protein with a viral envelope protein e.g. a glycoprotein, and as a result displayed on the surface of the particle.
Thus, the invention provides an HSV which is capable of targeting a specific cell type, said HSV lacking an expressible γ34.5 gene so as to lack neurovirulence and wherein the HSV expresses a targeting agent.
As an antibody binding domain forms a preferred embodiment of the invention, the following description will concentrate on the use of antibodies. However, it will be apparent to the skilled person that other targeting agents may be used, e.g. members of a specific binding pair such as a receptor and its ligand.
The antibody or antibody component incorporated into the viral envelope influences the selectivity of the virus by enhancing the efficiency of viral infection of a certain cell type or cell types. The HSV infection process is initiated through contact between glycoproteins of the viral envelope and glycoproteins of the target cell membrane. In the present invention, antibodies with specific affinity for membrane proteins of the chosen cell type are incorporated into the HSV viral envelope, increasing the affinity of the HSV for the surface of the chosen target cell through the additional interaction between the antibody and the cell surface protein. The binding of the antibody binding domain to its target antigen on the cell surface will bring both virion and cellular membranes into closer proximity and allow the viral envelope glycoproteins to initiate fusion of the membranes, leading to penetration of the cell.
The HSV-1 virion envelope contains at least 10 integral membrane glycoproteins, several of which mediate entry into mammalian cells. The initial interactions between the virus and the cell are between viral glycoproteins gB and/or gC and cell surface heparin sulphate proteoglycans, but this interaction is insufficient for viral penetration. Fusion of the viral and cellular membranes requires gD, gB and a gH/gL complex, and these proteins are presumed to act in concert. Specific receptor-mediated entry of HSV-1 involves interaction of gD with HVEM/HveA (herpesvirus entry mediator A), a lymphotoxin receptor and member of the TNF receptor family. Expression of HveA in CHO cells that are normally refractory to viral penetration rendered them permissive. Several other receptors have been identified using non-permissive CHO cells including the poliovirus receptor related proteins 1 and 2, now renamed HvecC and HvecB respectively. For this work, the inventors have been able to render a cell line normally resistant to infection permissive for HSV-1 entry.
The present invention uses an HSV that has an impaired ability to infect, replicate in or lyse terminally differentiated, non-dividing cells. In this form the virus is particularly suited for use as a therapeutic agent to treat diseases associated with proliferating cells such as cancer and non-cancer diseases such as Crohn's disease. The inventors believe the modified virus in accordance with the present invention will be particularly useful in targeting proliferating T-cells in cancer or non-cancer situations.
In a preferred embodiment, the HSV has been modified so that the gene encoding ICP 34.5 (gene γ34.5) is incapable of expressing a functional gene product.
The modified virus preferably contains a modification in respect of the wild type virus within the Bam HI s region of the internal repeat RL (0.81-0.83 mu) and within the counterpart region of the terminal RL (0-0.02 mu) such that the modified virus (variant) lacks neurovirulence.
The modification of the virus genome may be achieved by deletion of one or more nucleotides, insertion of additional nucleotides or any other alteration of the nucleotide sequence such as rearrangement, or substitution. Preferably, the modification of the HSV genome is achieved by deletion of one or more nucleotides.
The HSV may be a spontaneously isolated deletion variant of the wild type or it may be a wild type strain into which the desired modification has been introduced. Such modifications in the HSV may be made by genetic manipulation, for example by site-directed mutagenesis, or by excision of a portion of the genome with or without replacement with a pre-prepared DNA cassette incorporating the required modification.
Preferably, the HSV is HSV type I (HSV-I) even more preferably HSV-1 strain 17. In one embodiment, the HSV-1 strain will have a deletion of at least 100 nucleotides in the Bam HI s′ region between Alu I site at 125074 nb and 125972 nb and within its counterpart sequence in TRL.
More preferably 0.5 to 3 kb of the Bam HI s′ region and its counterpart in TRL is deleted. Still more preferably about 0.7-2.5 kb is deleted.
Suitable modified HSV include HSV-1 mutant 1716 or G207. The production of HSV1716 is described in EP 571,410-B which is incorporated herein by reference.
In addition to the above, the inventors have appreciated that, in order to treat a diverse range of tumours, the HSV complex in accordance with the invention will ideally have to be administered into the circulation of a patient. However, not only does the virus have to find the tumour cells (it can bind and be adsorbed by many different cell types) but it also has to contend with pre-existing immunological defenses (e.g. neutralising antibodies) designed to eliminate the virus. Pre-existing immunological defenses will be reasonably common as most people have had previous exposure to HSV-1. Given this, the present inventors have appreciated that there is a need to develop a “stealth” virus that avoids immunological detection and can specifically target tumour cells. Accordingly, the inventors have produced a stealth virus by eliminating the normal viral glycoproteins that mediate cell adsorption and replacing them with antibody-directed entry mediating glycoproteins incorporated into the virion structure. The principal viral glycoproteins involved in cell entry also provide the main neutralising epitopes and their removal will minimise immunological activity against such a virus. Thus, tumour antigen-directed HSV e.g. HSV1716 introduced into the circulation can target many tumour types including disseminated cancers that are either inaccessible or too numerous for direct injection or are too small to be detected.
Thus, the HSV complex as described above, e.g. HSV1716, that displays a tumour specific targeting antibody in accordance with the first aspect of the present invention, may be modified such that the genes encoding viral glycoproteins essential for normal cell entry (e.g. principally gD but also gC and/or gB, see below) are deleted or inactivated, thereby rendering the resulting virus dependent on tumour antigen/antibody interactions as the main route for cell infection. Deletion of these glycoproteins from the virus particle also removes the principal neutralising epitopes and therefore greatly reduces immunological defenses when administered systemically.
Therefore, the HSV complex according to the first aspect of the present invention may be further modified so that one or more viral glycoproteins, e.g. gD, gC and/or gB, are inactivated or deleted such that they are unable to mediate entry of the viral particle into cells. It is preferable that the one or more glycoproteins are modified at the genome level such that they cannot be expressed or cannot be expressed in a functional form. It is most preferable that the HSV genome is modified so that the one or more glycoproteins cannot be expressed at all as this will provide the HSV variant with the additional advantage that it can escape any pre-existing immunological defenses in vivo.
As mentioned above, the various glycoproteins may be modified or deleted from the viral particle, preferably at a nucleic acid level. The modification may include the incorporation of nucleic acid encoding the targeting agent so that the targeting agent is expressed on the surface of the particle. Thus, the HSV genome may be modified, in addition to the γ34.5 gene, such that one or more of the glycoproteins (e.g. gD, gC and/or gB) express the targeting agent, e.g. the antibody binding domain.
Preferably, the antibody or antibody component is specific for tumour surface antigen, i.e. antigen found on the surface of a tumour cell and associated with that cell, being either unique to tumour cells or being more abundant on tumour cells than on most if not all nontumour cells. Many novel or atypical forms of normal proteins are expressed by tumour cells, and antibodies directed against these provide tumour targeting strategies. For example, carcinoembryoinic antigen (CEA) is an important marker on many tumour cells and engineered antibodies directed against CEA have undergone clinical trials (Mayer, A. et al., J. Immunol. Methods 231 261-273 (1999)). Engineered antibodies directed against the Her2/neu growth factor (Trastuzamab) and against CD20 (rituximab) have been licensed for the treatment of breast cancer and Non-Hodgkin's lymphoma respectively Holliger, P. and Hoogenboom, H. (1998), Nature Biotechnology 16, 1015. CD55 (decay accelerating factor) is over-expressed by tumour cells to block complement activation and antibodies directed against CD55 may have therapeutic potential (Li, L. et al. B. J. Cancer 84 (1) 80-86 (2001)). Incorporation of an antibody binding domain that specifically targets tumour antigens such as CRA, Her2, CD20 and CD55 into the envelope of HSV will have the potential to alter its cellular tropism thus allowing infection of non-permissive tumour cells and possibly improving its ability to infect other tumour cells. For example, 1716 virions that display an antibody binding domain specific for CD20 may be able to infect and kill B-cell lymphomas.
Further, HSV, e.g. HSV1716 virions that display tumour targeting antibodies and from which the normal HSV-1 entry glycoproteins are deleted will only infect the targeted tumour cells.
The antibody binding domain may have specific affinity for a cell surface protein found on the cell type from which the tumour originated, e.g. in the case of a glioma, the antibody or antibody component incorporated into the HSV viral envelope would be specific for an antigen commonly associated with glial cells. The specificity of the avirulent HSV strain for infecting dividing cells would therefore be further modified so that glial cells were preferentially infected by the virus more than other types of dividing cells. By targeting dividing glial cells, the HSV should infect and lyse glioma cells more efficiently than any other cells. The use of antibodies or antibody components against particular cell types can also be used to extend the tropism of HSV to cell types that are not otherwise efficiently infected by HSV, e.g. the use of antibodies or antibody components specific for antigen found on B cells would be expected to extend the tropism of HSV to B cells. Antibodies or antibody components of different specificities may be included together in one HSV viral envelope. The combination of these specificities would be expected to give greater specificity of targeting to the desired cell type.
The antibody binding domain would preferably be fused to an integral membrane protein in the viral envelope, preferably an HSV glycoprotein. The preferred HSV-1 and HSV-2 glycoproteins are gB, gC and gD.
In a preferred embodiment, the antibody binding domain is in the form of a single chain variable fragment (scFv).
In a second aspect, the present invention comprises a method of making a modified HSV complex according to the first aspect of the invention comprising the step of infecting a cell line that constitutively expresses said fusion protein with an HSV, preferably a modified HSV, more preferably modified HSV-1.
In a third aspect, the present invention comprises a method of making a modified HSV complex according to the first aspect of the invention comprising the step of incorporating DNA encoding said fusion protein into the viral genome of the modified HSV.
In a fourth aspect, the present invention comprises the use of a modified HSV complex according to the first aspect of the invention in a method of medical treatment.
Preferably, the method comprises administering the HSV modified complex to a patient suffering from a disease associated with proliferation of cells, e.g. cancer, wherein the HSV complex selectively lyses dividing cells.
There is also provided a pharmaceutical composition which comprises the modified HSV in accordance with the present invention and a pharmaceutically acceptable carrier.
Pharmaceutical compositions comprising the modified HSV may be administered intravenously or directly injected into a tumour or infected site.